The THz-BRIDGE Project: The THz-BRIDGE Project: Tera-Hertz radiation in Biological Research, Tera-Hertz radiation in Biological Research, Investigation on Diagnostics and study on potential Genotoxic EffectsInvestigation on Diagnostics and study on potential Genotoxic Effects

RESEARCH OBJECTIVES OF THz-BRIDGE PROJECTRESEARCH OBJECTIVES OF THz-BRIDGE PROJECT

To provide a spectroscopic database for selected enzymes, proteins, biological membranes and cells in the frequency range from 100 GHz to 20 THz under irradiation conditions that preserve the integrity and functionality of the biological samples. (Workpackage 1)

To identify critical frequencies, which might induce damages on biological systems, and to determine the spectral regions for optimal contrast in imaging applications. (Workpackage 2)

To assess potential risks due to the exposure of membranes, cells, and DNA to pulsed and CW THz radiation and to define exposure standards for THz biomedical imaging applications; to monitor and recommend THz exposure conditions at specific occupational sites. (Workpackage 3)

The aim of this research project is to investigate the interaction of Terahertz (THz) radiation with biological systems. Present and rapidly increasing future applications of radiation in this spectral range, necessitate preventive rather than reactive research. The project follows a streamline of increasing complexity from bio-molecules to cells, e.g. membranes, chromosomal and DNA integrity. The objectives of the project are to analyze the physical mechanisms of interaction, to assess risk of potential damage to biological activity, both functional and morphological, and to guide and accompany the development of microscopic imaging at THz frequencies for biological and biomedical applications.

The project will bridge the existing gap of knowledge regarding the effects of Terahertz (THz) radiation on biological systems. THz radiation covers the frequency range between 100 GHz and 20 THz (i.e. a wavelength between 3 mm and 15 µm), which spans the spectral interval between the microwave- and the infrared regions of the electromagnetic spectrum.

Workplan – WP1

Development of biological samples, basic spectroscopic investigations inthe spectral range 100 GHz - 20 THzMeasurements of Reaction Induced Differential Spectra (RIDS)Time-resolved Spectroscopy at FEL Facilities“Time Domain Spectroscopy” (TDS) by solid state THz sourcesAssignment of absorption spectra to molecular bonds and functions

Workplan – WP 2

Study of the exposition of human blood to THz radiation as a function of: frequency, average and peak power, modulation conditions

Genotoxicity Test on human lymphocytes: Micronucleus, Comet, FISH Irradiation of DNA bases and measurement of the presence of fragments

by mass spectroscopy techniques Study of the exposure of liposomes to THz radiation: on-line evaluation of

the carbonic anhydrase (CA) activity under THz exposure as a function of frequency, average and peak power, and of modulation conditions

Investigations on epithelial cells: keratinocytes cell lines (NHK) and corneal cells (HCE-T) (Alamar-Blue cell cell viability assay) effects on the adhesion molecules of epithelial tissues (Fluorescein Leakage assay on tight junctions)

Modelling of the interaction mechanism

Workplan – WP 3

Spectroscopy of Proteins, Enzymes, Biological Membranes and Cells (Human Lymphocytes) : UFRANK, FZR-Dresden, USTUTT, ENEA, TVL

Evaluation of Biological Effects In Vitro DNA Bases and Human Lymphocytes: ICEmB, TAU, NHRF, ENEA, FZR-DresdenEvaluation of Biological Effects In Vitro: Membranes and epithelial-cell cultures: ICEmB, ENEA, UNOTT, FZR-Dresden, TVL

Safety issues at specific occupational sites: TVL, FZR-Dresden, UNOTT, TAU

Interdisciplinary consortium: 9 groups, 5 countries

Project start date: February 1, 2001Duration: 36 monthsTotal cost : 2 MEuro - EU Contribution : 1.4 MEuro

THz-BRIDGE PROJECT KEY FACTSTHz-BRIDGE PROJECT KEY FACTS

ENEA-Frascati (coordinator) (I) Forschungszentrum Rossendorf-Dresden (D) Tel-Aviv University - Dept. Physiology and Pharmacology (IL)Stuttgart University - I. Physics Institute (D)J.W. Goethe University - Biophysics Institute –Frankfurt (D)IREA -Naples, IMS-Rome, DIE-University of Rome - (ICEmB) (I)National Hellenic Research Foundation – Athens (EL)Teraview Limited – Cambridge (UK)University of Nottingham - Biomedical School (UK)

Whole blood samples are exposed to THz radiation under controlled conditions

After preparation of cultures, lymphocytes are induced to divide

After 44 h of growth Cytocalasin-B is added to stop cell division at nuclear level

Slide preparation and observation of Micronuclei frequency

EXAMPLE OF GENOTOXICITY TESTEXAMPLE OF GENOTOXICITY TESTTHE MICRONUCLEI ASSAITHE MICRONUCLEI ASSAI

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Statistical analysis of experimental results

EFFECTS ON MEMBRANE MODEL SYSTEMEFFECTS ON MEMBRANE MODEL SYSTEM

A good model for the membrane system is composed by carbonic-anydrase loaded liposomes vesicles. With such a model it is possible to evaluate the effects of THz radiation on the membrane permeability

The substrate has a very low self-diffusion rate across intact liposome bilayer. The substrate can be hydrolyzed by CA inside the liposomes only if it permeates

across the bilayer. Any radiation induced increase of hydrolysis rate of substrate catalyzed by CA

accounts for the effect on membrane permeability. The kinetic measurement is made following for 2-3 minutes the appearance of

reaction product p-nitrophenolate anion at its peak absorbance (=400 nm) on an Cary 50 Scan spectrophotometer

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I/I0 Phys. Sol. Serum Whole blood

Transmission of 120 GHz ENEA-FEL radiation was measured in saline solution, culture medium (not shown in the graph), serum and whole blood. Results were compared to water absorption

Culture medium = 83 cm-1

Saline Solution: = 79 cm-1

Whole blood: = 75 cm-1

Serum: = 71 cm-1

Water (non reported on the graph): = 80 cm-1

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47.6 cm-1 Uric Acid

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Human hemoglobin

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The THz absorption spectra of uric acid and human hemoglobin (Teraview Ltd.)

EXAMPLES OF SPECTROSCOPY MEASUREMENTSEXAMPLES OF SPECTROSCOPY MEASUREMENTS

Significant progress has been made on the design of spectroscopic cells and on the choice of suitable materials transparent in the frequency range of interest.

Polystyrene exhibits excellent optical properties in the wavelength range between 200 m and 3 mm (frequency range 1.5 THz to 100 GHz). Other materials like ZnTe can be used at shorter wavelengths.

Coherent radiation from Free Electron Lasers, Gas Lasers and Solid State Sources is available in a wide spectral range at the partner sites and at collaborating European FEL facilities.

Features: Wide tunability and power ranges, amplitude modulation Soft X-ray microscopy, SEM, Bio-labs available at ENEA, TAU and FZR

THE THz SOURCESTHE THz SOURCES

5 m

30 m 10 m

hBIAS P|E0(z,t)|2E0(z,t)

An ultrafast laser pulse is used to “switch” on a circuit (a coplanar transmission line antenna). A current transient is generated, producing a time-dependent dipole moment that emits radiation. The short duration of the pulse allows THz emission.

The non-linear properties of selected semiconductors are utilized to produce optical rectification of ultrashort laser pulses. The non linear polarization so generated travels in the medium with a lower phase velocity, due to the lattice vibration contribution. The result is an emission in a typical Cerenkov cone configuration.The rapid oscillations of the laser E field are rectified and only the envelop remains.

In a FREE ELECTRON LASER radiation is produced by a relativistic electron beam traveling through a magnetic structure (undulator). FELs can produce very short – high power THz laser pulse.

Auston SwitchAuston Switch Optical RectificationOptical Rectification Free Electron LaserFree Electron Laser

Survey of exposure conditions of technical personnel at specific occupational sites, where THz sources are employed or developed.

Distribution of a questionnaire to collect information

on the main radiation parameters of THz sources currently in use: (e.g. frequency range, power level, modulation)

on biological and potential biomedical applications of such sources

on the exposure conditions (if any) of technical personnel

on the safety measurements or precautions currently adopted Recommendations on safe exposure conditions.

Role of T-rays in the biomedical fieldRole of T-rays in the biomedical field

Biological applications are based on the specific spectroscopic fingerprints of biological matter in the THz spectral regions.

The different values of the absorption coefficient and index of refraction between water and tissue carbonated proteins at such frequencies, provide a unique contrast mechanism for biomedical imaging applications.

In the above picture, the THz image of basal cell carcinoma (BCC) obtained by Terahertz Pulse Imaging (TPITM) is shown on the left. The red areas indicate areas of cancer. On the right is the visible image showing no obvious signs of cancer growth.

Expected achievements and Expected achievements and preliminary results preliminary results

Spectroscopic measurements are used to identify the frequency intervals where THz radiation is absorbed by a specific system.

First spectroscopic investigations have been carried out on human blood, human hemoglobin, melanin and uric acid in the THz region.

An exposure set-up for the irradiation of human lymphocytes has been designed and constructed. First irradiation experiments on whole blood have been performed at a frequency of 120 GHz. Following irradiation, the Micronucleus assay was performed by ICEmB on human lymphocytes cultures.

The preliminary results obtained indicate that, under the adopted experimental conditions, THz radiation does not affect micronucleus frequency and cell cycle kinetics in peripheral blood human lymphocytes, as shown by comparing sham exposed cultures with exposed ones. Preliminary exposure of human skin cells to THz radiation has also been undertaken.

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FURTHER INFORMATIONFURTHER INFORMATION

This work has been carried out with financial support from the Commission of the European Communities, specific RTD programme “Quality of Life and Management of Living Resources”, Key Action 4 “Environment and Health” - contract QLK4-2000-00129.

Project ManagementEvaluation of results:

Spectral database & Reps.Risk Assessment

Safety Issues

Dissemination of results

Interface withInternational Projects

Identification of critical frequencies

Liposomes, blood & skin-cell cultures

WP-1Spectroscopy of proteins, enzymes

membranes and cells

WP-2Evaluation of biological effects

in vitro

WP-3Safety Issue at specific

Occupational sites

Spectral databaseon biological systems

Definition of exposure levelsSpectral information oncontrast in THz Imaging

Recommendations onTHz exposure

Assessment of THzradiation effects

An R&D project funded by the European Commission - 5th Framework Programme: Quality of Life and Management of Living Resources - Key Action 4-Environment and Health

Contract QLK4-CT2000-00129

Survey on the use of THz radiation on the THz-BRIDGE web site

THz-BRIDGE Web site http://www.frascati.enea.it/THz-BRIDGE

FREE ACCESS AREA Project Description Progress reports Official document Contacts

Project CoordinatorDr. G.P. Gallerano – ENEA FrascatiE-mail: gallerano@frascati.enea.itTel: +39-06-94005223 fax: +39-06-94005334

Typical Time Domain Spectroscopy layout

EU Scientific OfficerMinna Wilkki - EUROPEAN COMMISSIONE-mail: minna.wilkki@cem.eu.intTel: +32-2-299 5573fax: +32-2-296 4322